Heavy Metals, Oxidative Stress, and the Role of AhR Signaling

Abstract

The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcriptional factor pivotal in responding to environmental stress and maintaining cellular homeostasis. Exposure to specific xenobiotics or industrial compounds in the environment activates AhR and its subsequent signaling, inducing oxidative stress and related toxicity. Past research has also identified and characterized several classes of endogenous ligands, particularly some tryptophan (Trp) metabolic/catabolic products, that act as AhR agonists, influencing a variety of physiological and pathological states, including the modulation of immune responses and cell death. Heavy metals, being non-essential elements in the human body, are generally perceived as toxic and hazardous, originating either naturally or from industrial activities. Emerging evidence indicates that heavy metals significantly influence AhR activation and its downstream signaling. This review consolidates current knowledge on the modulation of the AhR signaling pathway by heavy metals, explores the consequences of co-exposure to AhR ligands and heavy metals, and investigates the interplay between oxidative stress and AhR activation, focusing on the regulation of immune responses and ferroptosis.

Introduction

The aryl hydrocarbon receptor (AhR) is a conserved, ligand-activated transcription factor belonging to the basic helix-loop-helix (bHLH)/Per-ARNT-Sim (PAS) family of proteins ¹. It features distinct domains that regulate its functions: the N-terminal bHLH domain is responsible for recognizing DNA binding sites, PAS domain A stabilizes heterodimerization with ARNT, and PAS domain B governs the binding pocket for ligands ². Early studies of AhR primarily centered on its toxicological aspects, as it was known to be activated by environmental pollutants such as dioxins ³. Upon AhR activation induced by these environmental xenobiotics, the metabolism of these substances results in the production of reactive oxygen species (ROS), which can disrupt cellular homeostasis over an extended period ⁴. Extensive research over the past two decades has investigated the regulatory relationship between AhR activation and oxidative stress ^5,6. As commonly known, heavy metal ions are strongly associated with the generation of reactive oxygen species (ROS) and subsequent oxidative toxicity upon cellular exposure ⁷. Therefore, the heavy metals-induced oxidative stress is highly implied to interact with AhR activation and its downstream target genes’ transcription ⁸. This concise review aims to provide a summary of the mechanisms through which various heavy metals activate the AhR signaling pathway and how heavy metals-induced oxidative stress can influence the regulation of AhR-related homeostasis.

AhR signaling and Its Ligands

In the canonical AhR signaling axis, AhR is released from a protein complex consisting of Src (SRC proto-oncogene, non-receptor tyrosine kinase), hsp90 (heat shock protein 90), p23 (PTGES3), and AhR-interacting protein (AIP) upon binding with an AhR ligand. This process exposes the nuclear localization signal of AhR, resulting in its nuclear translocation. In the nucleus, AhR dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT) and binds to xenobiotic response elements (XREs), initiating transactivation of targeting genes ^9–11. Among AhR targeted genes are Phase I metabolic enzymes including the cytochrome P450 superfamily proteins (CYPs), which introduce reactive and polar groups to xenobiotic molecules ¹². However, AhR also induces expression of target genes that function far beyond metabolism of xenobiotics ¹³. In fact, AhR plays a crucial role in development, metabolic homeostasis, and immune responses ^14–16, and it regulates expression of immunomodulatory proteins including programmed cell death 1 (PD-1), programmed death-ligand 1 (PD-L1), and indoleamine 2,3-dioxygenase 1 (IDO1) ^{11, 17}.

AhR activity is regulated by a wide range of small molecular compounds from the environment, diets, and metabolized by the host and its commensal microorganisms ^10,11,18,19. AhR was first identified and characterized through its activation by a diverse range of environmental ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) ²⁰. TCDD is a byproduct originating from the use of herbicides such as Agent Orange ²¹. It has been observed to persistently contaminate the soil and riverbed sediments over extended periods ²². PAHs are a group of organic compounds that are formed during incomplete combustion of organic materials such as fossil fuels, wood, and tobacco. Notably, benzo[a]pyrene (BaP) and dioxins are also known as pre-carcinogens or carcinogens. PCBs are a class of synthetic organic chemicals that are widely used in electrical equipment, hydraulic fluids, and other industrial applications, and PCBs can persist in the environment due to their long half-lives ²³. These environmental pollutant compounds are regarded as hazardous, toxic, and carcinogenic due to their ability to persist within the human body following exposure ^24,25. They are resistant to metabolic degradation, resulting in prolonged AhR activation ^26,27. This sustained activation disrupts the regulatory program of downstream genes, elevates oxidative stress levels, and disturbs overall homeostasis ²⁶.

Among the endogenous ligands for AhR are a series of compounds derived from tryptophan (Trp) metabolism/catabolism ^28–31, including 6-formylindolo[3,2-b] carbazole (FICZ), kynurenine (Kyn), indirubin, oxindole and indole 3-pyruvic acid (I3P). FICZ is converted from Trp either under UVB irradiation as a photoproduct or after exposure to H₂O₂ for 14 days at room temperature ^32,33; Kyn is derived from Trp by the enzyme metabolism of TDO (Tryptophan 2,3-dioxygenase)/IDO1 and arylformamidase (AFMID) ³⁰; Indirubin is a catabolic product of Trp that is metabolized by indole hydroxylation within the gut microbiota ³⁴. Oxindole, one of indole products, is primarily derived from Trp metabolism of gut microbiota, and its formation has been identified under cellular oxidation in our previous study ^35,36. I3P is a metabolic product of Trp catalyzed by IL4I1, an amino acid dioxygenase ³⁷.

Hence, an increasing number of studies have started to recognize Trp metabolites as indispensable factors in investigating the influence of AhR on cell survival, immune responses, and overall homeostasis.

AhR and Immune Checkpoint Genes

Immuno-modulators, or checkpoint proteins, are molecules that regulate the immune response, ensuring a balance between activation and suppression to maintain immune homeostasis ³⁸. IDO1 is a heme enzyme, which plays a critical role in regulating the tryptophan metabolism in the body, with its activity leading to the degradation of tryptophan and the production of kynurenine ³⁹. IDO-1 has been regarded as a third arm of the immune checkpoint ^40,41. Numerous prior studies have demonstrated that high expression of IDO1 is associated with an immunosuppressive effect, resulting in poor prognosis and outcomes in various solid tumors and hematologic malignancies ^42,43. The primary mechanism underlying the upregulation of IDO1 that contributes to immune tolerance is the inhibition of T cell activity in a tryptophan-depleting environment. The high expression of IDO1 in the tumor microenvironment depletes tryptophan, hindering T cells from utilizing it for self-activation ^30,44. Additionally, IDO1 induces the general control nonderepressible 2 (GCN2) kinase and suppresses the mammalian target of rapamycin 1 (mTOR1) pathway in T cells, thereby inhibiting their tryptophan-withdrawal functions ^45,46. The production of kynurenine, an AhR ligand, continually stimulates AhR activation, leading to increased IDO1 transcription and the formation of a positive feedback loop ⁴⁷. Moreover, AhR activation facilitates the trans differentiation of Th17 cells into regulatory T cells, further downregulating T cell activity ⁴⁸.

PD-1/PD-L1 are considered as major components of the immune checkpoint that negatively regulate T-cell functions ^49,50. PD-L1, also referred to as B7-H1 and CD274, is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells ⁵¹. The activation of PD-1/PD-L1 pathway serves to suppress autoimmunity and promote immune tolerance under inflammatory conditions ⁵². However, elevated expression of PD-L1 on tumors triggers an “adaptive immune mechanism” to evade anti-tumor responses and is strongly associated with advanced disease state and unfavorable prognosis in various cancers ⁵³. In earlier studies, AhR has been shown to regulate the transactivation of programmed cell death protein 1 (PD-1) and its ligand PD-L1, which are critical genes involved in immune suppression ^17,54,55. It has been reported that tobacco smoking carcinogens induced PD-L1 expression both in vivo and in vitro, which is highly dependent on AhR activation ⁵⁶. In a murine orthotopic oral cancer (MOC) model, AhR expression up-regulates T cell exhaustion in the tumor-infiltrating T cells and increases the expression of PD-1/PD-L1, while AhR-deficient model shows a lower PD-1/PD-L1 expression level and a long-lasting antitumor immune response ⁵⁴. Furthermore, the results obtained from both RT-PCR and NanoString PanCancer Immune Profiling Panel clearly demonstrated that AhR knockout significantly down-regulated the mRNA level of PD-L1 ⁵⁴. In T cells, Kyn induces expression of PD-1 through the AhR signaling axis, thus suppressing their cytotoxicity ¹⁷. In patients with gastrointestinal cancer, the immunomodulatory roles of AhR and its regulation of IDO1, PD-1/PD-L1 have also been identified. Strong evidence suggests that high expression of IDO1 and PD-L1 is associated with an early clinical stage and absence of lymphatic, which is AhR activation dependent ⁵⁷. Tryptophan 2,3-dioxygenase (TDO2), which has a similar function to IDO1, induces AhR-mediated PD-L1 transactivation to disrupt immune response ⁵⁸. Additionally, AhR/TDO2/kynurenine positive feedback loop facilitates liver metastasis of colon cancer via PD-L1-mediated immune evasion ⁵⁸. Given the crucial role of the immune checkpoint in cancer development, newly implemented immunotherapies targeting the blockage of IDO1 and/or PD-1/PD-L1 have better health outcomes of patients with various malignancies including melanoma, gastrointestinal cancer, breast cancer, and lung cancer. Although a phase III clinical trial failed to show an improved anti-tumor response with IDO1 inhibitor epacadostat ⁵⁹, it is possible that pro-survival activities of Trp-derivatives in the tumor microenvironment may greatly reduce its efficacy. Therefore, it’s worth considering the supplementary blockade of AhR activation as a viable strategy to enhance the anti-tumor efficacy of IDO1 inhibitors.

AhR and Oxidative Stress

The term “oxidative stress” refers to an elevation in the oxidation state within cells. This shift in cellular redox homeostasis typically occurs due to an increase in the production of reactive oxygen species and a failure of cellular antioxidant defenses ⁶⁰. Elevated ROS resulting from exposure to environmental toxins or disrupted metabolic processes can have detrimental effects on organisms ⁶¹. For example, tobacco smoke is a well-known ROS producer by generating hydrogen peroxide to disrupt antioxidant system ⁶². Benzo(a)pyrene, a well-known tobacco smoke byproduct, has been found to elevate ROS level in cells ⁵. Moreover, benzo(a)pyrene as an AhR activator is largely generated and consistently induce the activation of cytochrome P450 enzymes during smoking ⁶³.

Extensive research in the past has demonstrated that AhR activation plays a major role in the maintenance of oxidative homeostasis, which is accomplished through the regulation of xenobiotic metabolism, detoxification, and the modulation of antioxidant defenses ⁶⁴. Thus, dysregulated AhR activation can lead to oxidative stress and associated pathologies. Some researchers believe that when activated by TCDD, one of the most common environmental dioxins and an exogenous ligand of AhR, AhR may act as an inducer of oxidative stress ⁶⁵. The cytochrome P450 enzymes induced by TCDD are known to incompletely reduce O₂, resulting in the generation of superoxide and hydrogen peroxide due to poor coupling of electron flow ⁶⁶. TCDD has been implicated in the formation of the superoxide anion in rat brain with resultant generation of lipid peroxides, and this effect is controlled in part by the AhR complex ⁴. Additionally, TCDD has been shown to induce xanthine oxidase and xanthine dehydrogenase (XO/XDH) activities in the liver of mice ⁶⁷. This induction leads to the production of reactive oxygen species (ROS), which is dependent on AhR activation. An increase in ROS parameters is observed in liver when AhR downstream targets including cytochrome P450 (CYP) enzymes are activated to metabolize chemical compounds during the detoxification process ⁶⁸. One major rationale for AhR as an oncogene is that ROS accumulation due to increased CYP activities, leading to severe oxidative stress and increased DNA damage, favoring malignant transformation ^68,69. Mitochondria, as the major site of reactive oxygen production, also serves as a regulator of TCDD-induced oxidative stress. By treating mice with TCDD, succinate stimulated mitochondrial H₂O₂ production is elevated in the early weeks and remains significantly elevated after eight weeks. Meanwhile, the level of mitochondrial H₂O₂ production in AhR knock-out mice was one-fifth that found in wild-type mice ^70,71. Thus, a proposed mechanism by which TCDD-induced AhR activation contributes to oxidative stress is inhibition of electron transport at complex III, producing a persistent accumulation of succinate-dependent superoxide and H₂O₂ production in mitochondria ⁶⁶. Moreover, it has been reported that exposure to TCDD induced a degradation of mitochondria AhR (mitoAhR), which is localized to the inter-membrane space (IMS) of the organelle. The TCDD-induced degradation of mitoAhR significantly disrupted the cellular respiration and expression level of proteins within the mitochondrial proteome, resulting in mitochondrial dysfunction and TCDD-induced toxicity ⁷². Therefore, these experiments limit the relationship between AHR and ROS to the relationship where AHR is an inducer of ROS.

However, several lines of recent evidence suggest that ROS plays an essential role in activating AhR, thus enhancing expression of its downstream targets. In breast cancer cells, ROS accumulation is strongly correlated with increasing AhR expression, triggering AhR nuclear translocation, and promoting its transcriptional activity ⁷³. In addition, UVB irradiation or hydrogen peroxide treatment causes the accumulation of FICZ, which is mediated by inhibitory effect on CYP enzymes, thereby activating AhR ⁶. Furthermore, it has been demonstrated that H₂O₂ gradually transforms tryptophan into FICZ at room temperature ³³. This gradual conversion process produces nanomolar concentrations of FICZ, which are sufficient to induce AhR activation. In addition to the conversion of tryptophan to FICZ, kynurenine, as a typical AhR endogenous ligand, has also been identified as one of the degradation products resulting from tryptophan oxidation under ROS exposure ⁷⁴. In our previous finding, we identified 2-oxindole as a new compound generated in vitro during oxidative stress. 2-oxindole, also a Trp-derivative, was capable of activating AhR, leading to upregulation of CYP1A2, PD-L1, and IDO1 ³⁶. These lines of evidence strongly suggest that H₂O₂ and ROS can induce or accelerate the generation of AhR ligands in vivo.

Furthermore, recent research has uncovered a regulatory association between AhR and lipid peroxidation. While AhR’s metabolism induced by exogenous ligands can result in lipid peroxides, AhR plays a vital role in reducing levels of lipid peroxidation and providing resistance against ferroptosis. ⁷⁵. Ferroptosis is an iron-dependent cell death process, which is distinct from other forms of programmed cell death such as apoptosis, necrosis, and autophagy ⁷⁶. Ferroptotic cell death is marked by the presence of lipid species derived from peroxidation. Extensive research in the past has identified and characterized important gene products involved in regulating ferroptosis including those involved in iron metabolism (e.g., HO-1), anti-lipid peroxidation and anti-oxidation (e.g., GPX4), glutathione synthesis rate (e.g., SLC7A11, SLC3A2), and transcription factors ^77–81. Several lines of studies have implicated the involvement of AhR in modulating the sensitivity to ferroptosis. (I) AhR activation may regulate ferroptosis by modulating the expression of genes involved in ferroptotic responses. For example, AhR is capable of upregulating Nrf2 and its target gene, heme oxygenase-1 (HO-1) ^82,83. Both Nrf2 and HO-1 are known to exert antioxidant and cytoprotective effects, thus inhibiting ferroptosis. In addition, Kyn and I3P have been shown to suppress ferroptosis ^84,85. Since both Kyn and I3P are AhR agonists, it is conceivable that AhR and its signaling may potentiate ferroptotic responses. Furthermore, AhR may directly regulate expression of SLC7A11 to inhibit ferroptosis. Our studies revealed that the promoter region of SLC7A11 contains at least one XRE, a consensus binding motif of AhR, and AhR-deficiency suppresses expression of SLC7A11 after treatment with erastin (unpublished results).

Heavy Metal Toxicity and AhR

Heavy metal toxicity.

Heavy metals toxicity refers to the process by which certain metals or metalloids can elicit cellular and/or genetic alterations, leading to the dysfunction of physiological system, organ damage, and development of cancer ⁸⁶. One common feature associated with toxicity and/or carcinogenicity of metals/metalloids listed above is the perturbation of the oxidative state in the cells ⁸⁷. In fact, many toxic metals/metalloids induce the production of reactive oxygen species (ROS) through Fenton- and Haber-Weiss-type reactions, which are thought to be the main culprit in their carcinogenesis ⁸⁷.

Arsenic is a toxic metalloid that is abundant in the environment. It is generally thought that the resulting oxidative damage to cellular components is attributed to arsenic’s toxic effects and its role in carcinogenesis ⁸⁸. Arsenic exposure induces oxidative stress by promoting the generation of ROS and impairing cellular antioxidant defenses ⁸⁹. Furthermore, arsenic triggers significant morphological disruptions in mitochondrial integrity, accompanied by a decrease in mitochondrial membrane potential. These mitochondrial modifications are recognized as key loci where unregulated generation of superoxide anion radicals occurs ⁹⁰.

Chromium is a metallic element that exists in different oxidation states, with hexavalent chromium [Cr(VI)] being the most toxic and carcinogenic form ⁹¹. Acute exposure to Cr(VI) causes cytotoxicity and genotoxicity on cells, including the generation of ROS, perturbing cell signaling, induction of DNA double-strand breaks, and cell cycle arrest ^{92,93, 94,95}. Cr(VI)-generated ROS are important in causing various adverse health effects including mutagenesis and tumorigenesis. Under aerobic conditions, microsomal enzymes reduce Cr(VI) to Cr(V), which is accompanied by the generation of hydroxyl radical (HO^•) and superoxide (O₂^•) ⁹⁶. Superoxide (O₂^•) is converted to hydrogen peroxide (H₂O₂) by superoxide dismutase ⁹⁷. Therefore, Cr(VI) carcinogenesis is initiated or promoted by metabolic reduction of Cr(VI), producing ROS that can induce genotoxic effects, elicit inflammatory responses, and alter survival signaling pathways.

Cadmium is a heavy metal commonly found in the environment and it is classified as a human carcinogen, primarily associated with the development of lung cancer and prostate cancer ⁹⁸. Cadmium is known to promote oxidative stress, disrupting cellular signaling pathways, and perturbing cellular genetics ⁹⁹. Evidence showed that cadmium had a capacity to replace copper and iron from cytoplasmic and membrane proteins, including ferritin and apoferritin ¹⁰⁰. This cadmium-induced replacement results in an accumulation of unbound or inadequately chelated copper and iron ions, triggering oxidative stress through Fenton reactions ⁸⁷.

Lead, as a soft metal, has been wildly applied in the production of lead-acid batteries, oxides for paint, glass, and pigments ¹⁰¹. The primary organ system most susceptible to lead-induced toxicity is the central nervous system (CNS) ¹⁰². Additionally, both acute and chronic lead exposure can adversely affect the proper functioning of the kidney and reproductive organs ¹⁰². The toxicity of lead hinges on its disruption of the equilibrium between the production of ROS and the antioxidant defense mechanisms ¹⁰³. Lead shares electrons with sulfhydryl groups of antioxidant enzymes to establish covalent bonds, which inhibits their enzyme activities, leading to a depletion of glutathione ⁸⁶. Moreover, studies found that the mechanism of toxicity of lead also includes raising free radical levels and inducing lipid peroxidation ¹⁰⁴.

AhR activities impacted by heavy metals.

The connection between heavy metals and AhR activation has been the subject of thorough investigation in previous studies. Various hypotheses have been proposed to elucidate the mechanism underlying heavy metals induced AhR activation. However, despite differences in the effects of various heavy metals on AhR activation, a universally accepted explanation for the mechanism remains elusive.

Arsenic (As³⁺), presenting in elevated levels in drinking water and recognized as a potential human carcinogen, has demonstrated with the ability to induce AhR nuclear translocation and accumulation in Hepa-1 cells with a comparable efficacy to that of TCDD ¹⁰⁵. In addition, it has been found that As³⁺ exposure increase XRE-dependent luciferase activity and elevate CYP1A1 protein levels ¹⁰⁶ Meanwhile, As³⁺ have synergistic effect with BaP on phase1 enzyme CYP1A1, phase 2 enzyme NQO1 and potentiates AhR ligands-mediated stability of CYP1A1 mRNA ¹⁰⁷. However, the precise mechanisms underlying the ability of As³⁺ to induce CYP1A1 mRNA transcription via AhR activation remain unclear. One possible explanation is that As³⁺ increases HO-1 expression at the time, which facilitate heme degradation. This process generates biliverdin and subsequently bilirubin, which have been demonstrated to act as AhR ligands ¹⁰⁸. Nevertheless, despite the observed elevation of pulmonary CYP1A1 expression and an accompanying increase in total plasma bilirubin concentrations, administering bilirubin directly to the lungs through intra-tracheal injection failed to increase AhR activation and CYP1A1 mRNA expression ¹⁰⁹. The oxidative stress triggered by arsenic has been observed to liberate arachidonic acid from glycerophospholipids, potentially resulting in AhR activation ¹¹⁰. This could represent another plausible mechanism through which arsenic modulates the AhR signaling pathway.

As a ROS producer, Cr(VI) undergoes rapid reduction to chromium (V) and chromium (III) upon entering cells, which is accompanied by the generation of superoxide and H₂O₂ ^111,112. In our previous research, we discovered that Cr(VI)-induced ROS generation stimulates AhR activation ³⁶. The production of oxindole, a potent AhR ligand, is driven by the H₂O₂ generated because of Cr(VI) exposure ³⁶. The induction of AhR downstream genes by Cr(VI) exposure was inhibited by both AhR inhibitors and AhR genomic knockdown, which confirmed the regulation of AhR by Cr(VI) exposure ³⁶. Furthermore, we found that Cr(VI) exhibited synergy with I3P, an AhR ligand, in inducing the expression of CYP1A2 (unpublished data). Thus, we conclude that Cr(VI) regulates AhR activation by ROS generation.

Cadmium (Cd²⁺) is well known for its hepatic and renal toxicity ^113,114. Earlier studies have shown that the accumulation of Cd²⁺ affected total hepatic CYP450 enzymes activities in both in vitro/in vivo models ^115,116. In studies conducted in El-Kadi’s laboratory, Cd²⁺ was observed to enhance AhR nuclear translocation and elevate XRE-dependent luciferase activity, indicating that the induction of the CYP1A1 gene’s transcription by Cd²⁺ is dependent on the AhR signaling pathway ⁸. Moreover, Cd²⁺ was found to exhibit a synergistic effect on AhR activation whe n combined with AhR ligands or the pro-oxidant buthionine sulfoximine ⁷, suggesting that Cd²⁺ induces AhR activation by augmenting oxidative stress.

Lead (Pb²⁺) exposure has been shown to induce strong oxidative stress and antioxidant deficiency, leading to organs impairment and potential carcinogenesis ¹¹⁷. Initial investigations noted a substantial elevation in AhR levels upon exposure to Pb²⁺, accompanied with induction of the downstream gene CYP1A1 ¹¹⁸. In addition, Pb²⁺ enhanced the TCDD-induced increase in CYP1A1 mRNA levels in a manner dependent on both time and dosage ¹¹⁹. In research conducted in Korashy’s laboratory, lung toxicity has been associated with AhR activation and CYP1A1 induction induced by Pb²⁺ exposure ¹¹⁸. The initiation of inflammation and apoptosis in rat lung tissue by Pb²⁺ was asserted to rely on both oxidative stress and AhR activation ¹²⁰. Moreover, an elevation in the expression of CYP1A1 mRNA and protein levels was observed in Pb²⁺ induced cardiotoxicity, indicating a regulation role of AhR in cardiotoxicity ¹²¹. Co-exposure to BSO and Pb²⁺ enhanced CYP1A1 mRNA level, suggesting that Pb²⁺ regulate AhR signaling pathway through a mechanism dependent on oxidative stress ¹²².

Immuno-modulation upon Exposure to Heavy Metals

Early studies have provided a complex interaction between aberrant immune homeostasis and exposure to heavy metals while the mechanisms remain elusive. Previous studies have demonstrated that exposure to heavy metals can have diverse immune-related effects, encompassing the induction of proinflammatory cytokine dysregulation, hypersensitivity reactions, autoimmunity, immunosuppression, and disturbances in T-cell function ¹²³. For example, arsenic was found to regulate the activity of both innate and adaptive immunity by various cellular mechanisms. Chronic arsenic exposure markedly disrupted the differentiation process of peripheral blood monocytes into fully matured macrophages and inhibited the activity of transcriptional factors (such as NF-κB-related survival pathways and the liver X receptor), ultimately leading to a reduction in the expression of its target genes and disruption of cholesterol efflux ^124–126. Moreover, arsenic has been observed to significantly impair the activation and functionality of T cells, disrupting their differentiation and proliferation processes ¹²⁷. Long-term arsenic exposure induced reductions in lymphoproliferation, cytokine secretion, and an imbalance in Th1/Th2 and Th17/Treg differentiation, contributing to sever immunosuppression and immunotoxicity ^128,129.

Lead-induced immunotoxicity has been observed in both animal model experiments and occupational research studies ^130,131. Previous studies have reported that lead release elevated levels of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-12 in macrophages ¹³². In addition, a cohort study observed that lead-exposed workers exhibit higher plasma IL-10 levels and a tendency towards higher plasma TNF-alpha levels compared to non-exposed workers ¹³³. Lead has also been observed to reduce the percentage of CD4 cells and the expression of the CD4 surface antigen ^134,135. Furthermore, exposure to lead was reported to increase drug hypersensitivity syndrome (DHS) and sensitize food allergy by direct inhibition of antigen-specific T cell activation ^136,137.

Cadmium has been linked to numerous inflammatory manifestations and autoimmune responses across various tissues ¹²⁴. Cadmium-induced extensive ROS generation leads to the upregulation of several proinflammatory cytokines (such as IL-6, IL-8, TNF-α, and IL-1β) and inflammatory transcription factors like NF-κB, which facilitate additional leukocyte accumulation and contribute to a secondary injury ^138,139. In addition, evidence has been shown that AhR is involved in the lung leukocyte proinflammatory cytokine (IL-6) response to cadmium exposure ¹⁴⁰, suggesting an interaction between AhR and heavy metals modulated immune response.

It has been known that AhR plays an important role in both innate and adaptive immunity, partly through mediating expression of immune checkpoint proteins including IDO1, PD-1 and PD-L1 ^54,56,141. Our previous studies have shown that H₂O₂ and ROS generated AhR ligand oxindole significantly induced IDO1 and PD-L1 expression in both concentration- and time-dependent fashion ³⁶. Given the fact that heavy metals regulate AhR by ROS generation, we propose a novel hypothesis to elucidate the mechanism linking heavy metals and immune regulation. This hypothesis posits that heavy metals regulate immune checkpoint genes through the generation of ROS, thereby inducing the production of endogenous AhR ligands, particularly oxindole. This, in turn, leads to an increase in the expression of immune checkpoint genes such as IDO1 and PD-L1 through an AhR activation-dependent mechanism. Given this hypothesis, we demonstrated that Cr(VI) induced expression of PD-L1 and IDO1 in HCT116 cells in a concentration- and time-dependent manner (unpublished data). Combined with the above findings strongly suggest that heavy metals potentially function as modulator of immune checkpoint genes in vivo and that the heavy metals induced oxidative stress may significantly compromise immune responses via deregulating AhR signaling.

Ferroptotic Responses impacted by Heavy Metals

As mentioned earlier, heavy metals can stimulate the generation of ROS, which react with membrane polyunsaturated fatty acids (PUFAs) to generate lipid peroxides, ultimately triggering lipid peroxidation ^142,143. Pioneering research has recently provided compelling evidence linking heavy metal-induced lipid peroxidation to ferroptosis, a process closely associated with metal toxicity ¹⁴⁴. Chronic As³⁺ exposure has been found to trigger ferroptosis in a mice model, with mechanisms involving iron accumulation, disruption of mitochondrial function, endoplasmic reticulum stress and significant reductions in anti-ferroptosis protein expressions (such as SLC7A11, GPX4), ultimately resulting in cerebral cortex neuronal loss and cell death within the testicular tissue ^145,146. Additional evidence has revealed that arsenic exposure induce mitochondrial reactive oxygen species (MtROS)-dependent autophagy in pancreatic cells, leading to ferroptosis by modulating iron homeostasis ¹⁴⁷. Furthermore, arsenic exposure has been revealed to upregulate ACSL4 (acyl-CoA synthetase long-chain family member 4) under ferroptosis ¹⁴⁸. The inhibition of ACSL4 has been shown to significantly suppress ferroptosis, inflammation, and lipid accumulation triggered by arsenic exposure, highlighting ACSL4 as a promising new target for anti-ferroptotic strategies ¹⁴⁸.

Recent studies have observed that Cd induced liver damage by inducing ferroptosis ¹⁴⁹. One mechanism by which Cd induces ferroptosis is through the augmentation of endoplasmic reticulum (ER) stress, accompanied by the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway ¹⁴⁹. Moreover, exposure to Cd led to an elevation in cellular iron content and oxidative stress, resulting in the upregulation of iron death-related genes (such as ASCL4, PTGS2, and NOX1) expression, while concurrently repressing the expression of anti-ferroptosis genes (like GPX4 and FTH1) ¹⁵⁰. Intriguingly, contrary to its role as a ferroptosis inducer, chronic exposure to Cd was discovered to inhibit erastin-induced ferroptosis by reducing cellular iron content, elevating GSH levels, and upregulating SLC7A11 expression in prostate cancer cells ^151,152. The anti-ferroptosis function of Cd increases cancer cell viability via a possible modulation of miR-128-3p/SLC7A11 signaling ¹⁵¹. This finding suggests a dual-edged effect of heavy metals in ferroptosis, which depends on both the duration and dosage of exposure.

Compelling studies have revealed a connection between lead-induced toxicity in the central nervous system (CNS) and ferroptosis. Utilizing the ferroptosis database for bioinformatics analysis, it was discovered that 16 ferroptosis-related genes exhibited differential expression in choroid plexus epithelial cells after exposure to lead ¹⁵³. Furthermore, Pb²⁺ significantly inhibited GPX4 and SLC7A11 expression level and decreased cell viability, leading to massive ferroptosis and a potential blood-cerebrospinal fluid barrier dysfunction ¹⁵³. Ferroptosis-induced lead neurotoxicity has been observed in HT22 cells. Acute exposure to lead resulted in the accumulation of iron, a reduction in glutathione (GSH), and a significant downregulation of the expression of SLC7A11 ¹⁵⁴. These studies imply that SLC7A11 plays as an important regulator of lead induced ferroptosis.

Even though Cr(VI) is known to induce oxidative stress and high dose Cr(VI) exposure directly contribute to cell death, we observed that at low concentrations, Cr(VI) suppressed erastin-induced ferroptosis and cell death (unpublished data). Given our observations that Cr(VI) activates AhR signaling ³⁶, we hypothesized that Cr(VI) suppress ferroptosis induced by erastin by inducing AhR activation, with mechanism involving a positive feedback loop between AhR, its transcriptional targets (e.g., IDO1), and metabolic products (e.g., Kyn) catalyzed by these enzymes. In addition, we observed that AhR-null cells, as originally described ¹⁵⁵, contained a higher basal level of HO-1 than wild-type cells and erastin treatment further increased expression of HO-1(unpublished data). This observation suggests that AhR may be directly involved in the negative regulation of HO-1 during ferroptosis. It is known that heme degradation by HO-1 leads to the release of free iron, which can participate in the Fenton reaction, resulting in highly reactive hydroxyl radicals ¹⁵⁶. The increased level of these radicals is expected to contribute to lipid peroxidation, a key feature of ferroptosis. Thus, this line of observation introduces a novel perspective to elucidate how heavy metals modulate ferroptosis through an AhR-dependent pathway.

Summary

In this review, we discuss the intricate relationship between oxidative stress and the AhR signaling pathway, particularly under the influence of heavy metal exposure, such as [Cr(VI). Recent findings underscore that AhR not only modulates oxidative stress but also experiences reciprocal regulation through stress-induced endogenous ligands. Specifically, oxidative stress can trigger the formation of metabolites from cellular components, like amino acids and lipids, that serve as AhR agonists.

Our proposed model, illustrated in Figure 1, outlines the activation of AhR signaling by oxidative stress, which is a critical response to environmental metal toxicants, including cadmium, arsenic, lead, and chromium. These metals/metalloids induce the production of ROS, leading to the generation of amino acid-derived compounds such as 2-oxindole, Kyn, and I3P. These substances act in turn as AhR ligands, causing the receptor to translocate to the nucleus and bind to XRE to regulate the transcription of target genes. Upon activation, AhR transactivates expression of genes related to ferroptosis, such as SLC7A11 and heme oxygenase 1 (HO-1), and immuno-modulatory responses, such as IDO1 and PD-L1.

Figure 1:

AhR activation under oxidative stress: This model illustrates the process of AhR activation in response to oxidative stress, involving the generation of Trp-derivatives. Upon activation, AhR plays a crucial role in the transcriptional regulation of target genes. This activation has significant biological consequences, including the suppression of immune responses and ferroptosis, providing insights into the complex interplay between AhR signaling and cellular stress responses.

In this comprehensive review, we explore the complex interaction between various heavy metals and the AhR signaling pathway. We place particular emphasis on highlighting oxidative stress as a key mechanism underlying heavy metal-induced AhR activation. Additionally, we provide an extensive overview of the regulatory interaction between AhR and heavy metals and their impact on immune responses and ferroptosis. Furthermore, we propose a novel hypothesis suggesting that the regulation of immune responses and ferroptosis by heavy metals may be dependent on AhR activation. This novel perspective may contribute to unraveling the previously unknown mechanisms by which heavy metals may exert impact on immune responses and ferroptosis via the AhR signaling axis.

Highlights:

1. Oxidative stress as a key mechanism underlying heavy metal-induced AhR activation.

2. AhR activation is intricately linked to oxidative stress, immune regulation, and the induction of ferroptosis.